Production of Recombinant Proteins

Novel Microbial and Eukaryotic Expression Systems

Edited byGerd Gellissen

InnodataFile Attachment3527604413.jpg

Production of Recombinant Proteins

Edited byGerd Gellissen

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Production of Recombinant Proteins

Novel Microbial and Eukaryotic Expression Systems

Edited byGerd Gellissen

Edited by

Prof. Dr.Gerd GellissenRingstrasse 3042489 WülfrathGermany

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This book is dedicated to my wife Gabiand my sons Benedikt, Georg, and Ulrich.

Preface

Gene technology has invaded the production of proteins, and especially productionprocesses for pharmaceuticals. At the beginning of this new technology only a limitednumber of microorganisms was employed for such processes, namely the bacteriumEscherichia coli, followed by the baker’s yeast Saccharomyces cerevisiae as a microbialeukaryote. For both organisms a wealth of information was available which stemmedfrom a long tradition of safe use in science and, in case of the yeast, also from foodmanufacturing. However, certain limitations and restrictions urged the search foralternatives that were able to meet the requirements and demands for the expressionof an ever-growing number of target genes. As a consequence, a plethora of microbialand cellular expression platforms were developed. Nonetheless, the range of launchedproducts still leans for the most part on production in a restricted set of organisms,with most of the newly identified microbes being applied to research in academia.

Despite superior characteristics of some industrially employed platforms, limita-tions and restrictions are still encountered in particular process developments. In apublicly funded program, Rhein Biotech has set out with academic partners in therecent past to identify additional microbes with attractive capabilities that could sup-plement its key system, Hansenula polymorpha. As such, the Gram-positive Staphylo-coccus carnosus, the thermo- and osmotolerant dimorphic yeast species Arxula adeni-nivorans, the filamentous fungi Aspergillus sojae, and the nonsporulating species Sor-daria macrospora, were developed. This development was supplemented by toolssuch as the definition of fermentation conditions and a “universal vector” that canbe employed to target a range of fungi for the identification of the most suited plat-form in particular process developments. The application of these platforms andtools is included in the business concept of a new German biotech start-up company,MedArtis Pharmaceuticals GmbH, Aachen.

The present book is aimed at providing a comprehensive view of these newly iden-tified and defined systems, and comparing them with a range of established andnew alternatives. The book includes the description of two Gram-negative organisms(E. coli and Pseudomonas fluorescens), the Gram-positive Staphylococcus carnosus, fouryeast species (Arxula adeninivorans, Hansenula polymorpha, Pichia pastoris and Yarro-wia lipolytica), and the two filamentous fungi Aspergillus sojae and Sordaria macro-spora. The description of these microbial platforms is further supplemented by anoverview on expression in mammalian and plant cells.

VII

I would like to thank all academic partners who co-operated in the development ofthese new platforms. I gratefully acknowledge funding by the Ministry of EconomyNRW, Germany (TPW-9910v08). I would also like to thank D. Ellens, M. Piontek,and F. Ubags, who inspired me to edit this book.

I also express my gratitude to all authors for their fine efforts and contributions,and thank Dr. Paul Hardy, Düsseldorf, for carefully reading some of the manu-scripts. I also acknowledge the continuous support of Dr. A. Pillmann and her staffat Wiley-VCH.

Aachen, October 2004 Gerd Gellissen

VIII Preface

Foreword

The availability of ever-increasing numbers of eukaryotic, prokaryotic, and viral gen-omes facilitates the rapid identification, amplification, and cloning of coding se-quences for technical enzymes and pharmaceuticals, including vaccines. To take advan-tage of the treasures of information contained in these sequences, elegant multiplat-form expression systems are needed that fulfill the specific requirements demanded byeach potential application; for example, economy in the case of technical enzyme pro-duction, or safety and authenticity in the case of pharmaceutical production.Therefore,while Escherichia coli and other bacteria may be perfectly suited for technical enzymeproduction or the production of selected pharmaceuticals requiring no special modifi-cation, eukaryotic organisms may be advisable for applications where safety (e.g., noendotoxin), contamination, or authenticity (e.g., proper protein modification by glyco-sylation) are of concern. While the choices of microbial and eukaryotic expression sys-tems for the production of recombinant proteins are many in number, most research-ers in academic and industrial settings do not have ready access to pertinent biologicaland technical information as it is usually scattered in the scientific literature. This bookaims to close this gap by providing, in each chapter, information on the general biologyof the host organism, a description of the expression platform, a methodological sec-tion (with strains, genetic elements, vectors and special methods, where applicable),and finally some examples of proteins expressed with the respective platform. The de-scribed systems are well balanced by including three prokaryotes (two Gram-negativeand one Gram-positive), four yeasts, two filamentous fungi, and two higher eukaryoticcell systems (mammalian and plant cells). The book is rounded off by providing valu-able practical and theoretical information about criteria and schemes for selection ofthe appropriate expression platform, about the possibility and practicality of a universalexpression vector, and about comparative industrial-scale fermentation. The produc-tion of a recombinant Hepatitis B vaccine is chosen to illustrate an industrial example.As a whole, this book is a valuable and overdue resource for a varied audience. It is apractical guide for academic and industrial researchers who are confronted with thedesign of the most suitable expression platform for their favorite protein for technicalor pharmaceutical purposes. In addition, the book is also a valuable study resource forprofessors and students in the fields of applied biology and biotechnology.

Fort Collins, Colorado, U.S.A., June 2004 Herbert P. Schweizer, Ph.D.

IX

Contents

1 Key and Criteria to the Selection of an Expression Platform 1Gerd Gellissen, Alexander W.M. Strasser, and Manfred Suckow

2 Escherichia coli 7Josef Altenbuchner and Ralf Mattes

2.1 Introduction 82.2 Strains, Genome, and Cultivation 92.3 Expression Vectors 112.3.1 Replication of pMB1-derived Vectors 112.3.2 Plasmid Partitioning 112.3.3 Genome Engineering 132.3.4 E. coli Promoters 142.4 Regulation of Gene Expression 152.4.1 Negative Control 162.4.2 Positive Control 182.4.2.1 l-Arabinose Operon 182.4.2.2 l-Rhamnose Operon 192.5 Transcription and Translation 212.5.1 Translation Initiation 212.5.2 Codon Usage 222.5.3 Translation Termination 262.5.4 Transcription Termination and mRNA Stability 262.6 Protein Production 272.6.1 Inclusion Body Formation 272.6.1.1 Chaperones as Facilitators of Folding 282.6.1.2 Fusion Protein Technology 292.6.2 Methionine Processing 292.6.3 Secretion into the Periplasm 302.6.4 Disulfide Bond Formation and Folding 312.6.5 Twin Arginine Translocation (TAT) of Folded Proteins 312.6.6 Disulfide Bond Formation in the Cytoplasm 322.6.7 Cell Surface Display and Secretion across the Outer Membrane 332.7 Examples of Products and Processes 34

XI

2.8 Conclusions and Future Perspectives 35Appendix 36References 37

3 Pseudomonas fluorescens 45Lawrence C. Chew, Tom M. Ramseier, Diane M. Retallack,Jane C. Schneider, Charles H. Squires, and Henry W. Talbot

3.1 Introduction 453.2 Biology of Pseudomonas f luorescens 473.3 History and Taxonomy of Pseudomonas f luorescens Strain Biovar I

MB101 473.4 Cultivation 483.5 Genomics and Functional Genomics of P. f luorescens Strain MB101 493.6 Core Expression Platform for Heterologous Proteins 523.6.1 Antibiotic-free Plasmids using pyrF and proC 523.6.2 Gene Deletion Strategy and Re-usable Markers 533.6.3 Periplasmic Secretion and Use of Transposomes 543.6.4 Alternative Expression Systems: Anthranilate and Benzoate-inducible

Promoters 543.7 Production of Heterologous Proteins in P. f luorescens 553.7.1 Pharmaceutical Proteins 553.7.2 Industrial Enzymes 593.7.3 Agricultural Proteins 603.8 Conclusions 60

Appendix 61References 62

4 Staphylococcus carnosus and other Gram-positive Bacteria 67Roland Freudl

4.1 Introduction 674.2 Major Protein Export Routes in Gram-positive Bacteria 684.2.1 The General Secretion (Sec) Pathway 694.2.2 The Twin-Arginine Translocation (Tat) Pathway 714.2.3 Secretion Signals 724.3 Extracytosolic Protein Folding 734.4 The Cell Wall as a Barrier for the Secretion of Heterologous

Proteins 754.5 Degradation of Exported Proteins by Cell-associated and Secreted

Proteases 754.6 Staphylococcus carnosus 764.6.1 General Description 764.6.2 Microbiological and Molecular Biological Tools 774.6.3 S. carnosus as Host Organism for the Analysis of Staphylococcal-related

Pathogenicity Aspects 774.6.4 Secretory Production of Heterologous Proteins by S. carnosus 78

XII Contents

4.6.4.1 The Staphylococcus hyicus Lipase: Secretory Signals and HeterologousExpression in S. carnosus 78

4.6.4.2 Use of the Pre-pro-part of the S. hyicus Lipase for the Secretion ofHeterologous Proteins in S. carnosus 80

4.6.4.3 Process Development for the Secretory Production of a Human CalcitoninPrecursor Fusion Protein by S. carnosus 81

4.6.5 Surface Display on S. carnosus 82Appendix 83References 84

5 Arxula adeninivorans 89Erik Böer, Gerd Gellissen, and Gotthard Kunze

5.1 History of A. adeninivorans Research 895.2 Physiology and Temperature-dependent Dimorphism 915.3 Genetics and Molecular Biology 965.4 Arxula adeninivorans as a Gene Donor 975.5 The A. adeninivorans-based Platform 995.5.1 Transformation System 995.5.2 Heterologous Gene Expression 995.6 Conclusions and Perspectives 105

Acknowledgments 105Appendix 105References 108

6 Hansenula polymorpha 111Hyun Ah Kang and Gerd Gellissen

6.1 History, Phylogenetic Position, Basic Genetics and Biochemistry ofH. polymorpha 112

6.2 Characteristics of the H. polymorpha Genome 1156.3 N-linked glycosylation in H. polymorpha 1186.4 The H. polymorpha-based Expression Platform 1206.4.1 Transformation 1206.4.2 Strains 1226.4.3 Plasmids and Available Elements 1246.5 Product and Process Examples 1276.6 Future Directions and Conclusion 1296.6.1 Limitations of the H. polymorpha-based Expression Platform 1296.6.2 Impact of Functional Genomics on Development of the H. polymorpha

RB11-based Expression Platform 130Appendix 132References 136

7 Pichia pastoris 143Christine Ilgen, Joan Lin-Cereghino, and James M. Cregg

7.1 Introduction 143

XIIIContents